JP6543849B2 - Observation device - Google Patents

Description

  The present invention relates to an observation device.

  Non-Patent Document 1 discloses a two-photon microscope. The two-photon microscope of Non-Patent Document 1 irradiates the brain of a mouse with laser light to be excitation light. And he observes various areas of the brain. Further, Non-Patent Document 2 discloses a multiphoton microscope. The multiphoton microscope of Non Patent Literature 2 is a rotary microscope that can rotate an objective lens in a range of -5 ° to + 95 °.

Sofroniew et al., “A large field of view two-photon mesoscope with subcellular resolution for in vivo imaging” eLife 2016; 5: e14472. https://www.thorlabs.co.jp/newgrouppage9.cfm?objectgroup_id=7494&pn=bergamo#ad-image-0: Internet March 23, 2017 search

  In brain research, it is desirable to observe various parts from various directions for wide area network observation. However, in the microscope of Non-Patent Document 2, the objective lens can be rotated only around one axis. Thus, in order to observe from various directions, it is necessary to rotate the sample. However, when the sample is an experimental animal such as a mouse or a marmoset, when the sample is rotated, the sample may be affected by stress or the like. Therefore, there is a problem that it is difficult to observe from various directions without being affected by the device.

  The present invention has been made on the background of such a situation, and an object of the present invention is to provide an observation device capable of observing a sample from various directions.

  An observation apparatus according to an aspect of the present embodiment includes a rotation shaft rotatably provided about a first axis, a rotation axis drive mechanism for rotating the rotation axis about the first axis, and the rotation axis An arm provided with a first guide mechanism, and an optical system unit movably provided along the first guide mechanism, the irradiation light being irradiated to the sample An optical system unit having a scanner for scanning the object, an objective lens attached to the sample side of the optical system unit, for condensing the irradiation light scanned by the scanner onto the sample, and the objective lens of the first axis And a unit drive mechanism for moving the optical system unit along the first guide mechanism so as to rotationally move around a rotation center point on an extension line.

  In the above observation apparatus, the rotation axis is hollow so that light can pass through the inside of the rotation axis, and the observation apparatus is attached to the arm on the first axis, and the rotation axis A movable mirror that reflects the irradiation light that has passed through the inside of the lens, a movable mirror that is provided movably with respect to the arm, and reflects the irradiation light from the rotation mirror toward the optical system unit; The apparatus may further include a mirror drive mechanism that moves the movable mirror so as to change the distance between the rotating mirror and the movable mirror in accordance with the position of the optical system unit in the guide mechanism.

  In the above observation apparatus, the arm may be provided with a second guide mechanism that guides the rectilinear movement of the movable mirror by the mirror drive mechanism.

  In the above-described observation apparatus, the observation device includes a first angle variable mirror provided in the optical system unit to which the irradiation light reflected by the movable mirror is incident, and the irradiation light reflected by the first angle variable mirror is transmitted by the scanner The scanned angle of the first variable angle mirror may be changed according to the position of the optical system unit in the first guide mechanism.

  In the above observation apparatus, the rotation axis is hollow so that light can pass through the inside of the rotation axis, and the observation apparatus is attached to the arm on the first axis, and the rotation axis And a second variable-angle mirror provided on the arm to reflect light emitted from the rotating mirror toward the optical system unit, the optical The system unit is attached to the optical system unit and reflects a third angle variable mirror that reflects the irradiation light reflected by the second angle variable mirror, and reflects the irradiation light reflected by the third angle variable mirror And the angle of the third variable angle mirror is changed according to the incident position of the irradiation light to the mirror, and the irradiation light is incident on the third variable angle mirror. Depending on the location, angle of the second variable angle mirrors may be changed.

  In the above observation apparatus, the third variable angle mirror and the mirror are respectively provided on a substrate, a front surface side of the substrate, and a reflective film formed to transmit a part of the irradiation light. A position sensor provided on the back side of the substrate to detect the incident position of the irradiation light, and the third variable angle mirror according to the incident position detected by the position sensor of the mirror The angle may be changed, and the angle of the second variable-angle mirror may be changed according to the incident position detected by the position sensor of the third variable-angle mirror.

  In the above observation apparatus, a first beam sampler disposed in an optical path between the third variable angle mirror and the mirror and extracting a part of the irradiation light reflected by the third variable angle mirror. A first position sensor for detecting an incident position of the irradiation light extracted by the first beam sampler; and an optical path disposed between the mirror and the scanner and reflected by the mirror It further comprises a second beam sampler for taking out a part, and a second position sensor for detecting an incident position of the irradiation light taken out by the first beam sampler, and is detected by the first position sensor. The angle of the second variable angle mirror is changed according to the incident position, and the angle of the third variable angle mirror is changed according to the incident position detected by the second position sensor. The Good.

  The above observation apparatus may further include a pulse laser light source that generates the irradiation light.

  The above observation apparatus may further include an optical fiber for introducing the irradiation light into the optical system unit.

  In the above observation apparatus, a counter weight may be attached to the arm at a position symmetrical to the optical system unit with the rotation center point interposed therebetween.

  According to the present invention, it is possible to provide an observation device capable of observing a sample from various directions.

FIG. 2 is a perspective view showing the configuration of the main part of the observation apparatus according to the first embodiment. FIG. 2 is an XY plan view showing the configuration of the main part of the observation device according to Embodiment 1. FIG. 7 is a YZ plan view showing the operation of the observation device according to Embodiment 1. FIG. 10 is a perspective view showing the configuration of the main part of the observation device according to Embodiment 2. FIG. 14 is a YZ plan view showing the operation of the observation device according to the second embodiment. FIG. 7 is a diagram showing the configuration of an optical system of an observation apparatus according to a second embodiment. FIG. 7 is a diagram showing the configuration of an optical system of an observation apparatus according to a second embodiment. It is a figure for demonstrating relationship with (theta) angle and the angle correction mirror of a movable mirror. FIG. 10 is a control block diagram of the observation device according to Embodiment 2. FIG. 14 is a diagram showing an operation of the observation device according to the third embodiment. FIG. 16 is a diagram showing the configuration of a position sensor mirror used in the observation apparatus according to the third embodiment. FIG. 16 is a diagram for explaining a servo loop used in the observation device according to the third embodiment. FIG. 18 is a diagram for describing a configuration of an observation device according to a first modification of the third embodiment.

  Hereinafter, embodiments to which the present invention can be applied will be described. The following description is for describing the embodiments of the present invention, and the present invention is not limited to the following embodiments. The following description is omitted and simplified as appropriate for clarification of the explanation. In addition, one skilled in the art would be able to easily change, add, and convert the elements of the following embodiments within the scope of the present invention. In addition, what attached | subjected the same code | symbol in each figure has shown the same element, and description is abbreviate | omitted suitably.

Embodiment 1
The configuration of the main part of the observation apparatus according to the present embodiment will be described using FIG. 1 and FIG. FIG. 1 is a perspective view showing the configuration of the main part of the observation device 100. As shown in FIG. Specifically, FIG. 1 schematically shows the configuration of a holding device 10 that rotatably holds the optical system unit 110 and the objective lens 120 of the observation apparatus 100. The holding device 10 rotatably holds a part of the optical system in order to observe the sample from various directions without moving the sample. FIG. 2 is an XY plan view showing the configuration of the holding device 10. In addition, the one part structure is suitably abbreviate | omitted in FIG. 1, FIG. For example, the illumination light source and the like provided in the observation device 100 are omitted.

  The observation apparatus 100 includes a rotating shaft 11, an arm 12, a first weight 13, a second weight 14, a guide mechanism 15, a bearing 16, a pedestal 17, an optical system unit 110, and an objective lens 120. And have. The following description will be made using an XYZ three-dimensional orthogonal coordinate system as appropriate. The Z axis is a central axis of the rotation axis 11, and is in the horizontal direction here. Also, the X direction is a horizontal direction, and the Y direction is a vertical direction. Of course, the X-axis, the Y-axis, and the Z-axis are relative, and change according to the installation direction of the observation apparatus 100. A point at which the optical axis OX of the objective lens 120 intersects with the Z axis is taken as an observation center point O. A sample (not shown in FIGS. 1 and 2) is disposed at the observation center point O. The sample is placed on a sample stage (not shown).

  The rotation axis 11 is provided along the Z direction. The rotating shaft 11 is cylindrical or cylindrical. Specifically, the Z axis coincides with the rotation center of the rotation shaft 11. The rotating shaft 11 is fixed to a pedestal 17 (not shown in FIG. 1) via a bearing 16. That is, the pedestal 17 and the bearing 16 rotatably hold the rotating shaft 11. The rotation axis 11 rotates around the Z axis. As shown in FIG. 1, the rotation direction around the Z axis is taken as the φ direction. Also, rotation in the φ direction is also referred to as φ-axis rotation.

  An arm 12 is attached to one end of the rotating shaft 11. The arm 12 is connected to the tip of the rotating shaft 11. The arm 12 is a horseshoe-shaped plate extending in two directions from the rotation shaft 11. The central portion of the arm 12 is fixed to the rotating shaft 11, and both ends extend in the -Z direction. The shape of the arm 12 is not particularly limited, and may be C-shaped or U-shaped. The arm 12 rotates in the φ direction with the rotation shaft 11. That is, when the rotary shaft 11 is rotated by a drive mechanism such as a motor, the arm 12 rotates in the φ direction.

  As shown in FIGS. 1 and 2, an angle at which the arm 12 is in parallel with the XY plane is taken as a reference angle of φ axis rotation. At the reference angle, one end of the arm 12 is disposed above the rotating shaft 11 (+ Y side), and the other end is disposed below the Z axis (−Y side). The rotation shaft 11 can rotate, for example, ± 30 ° in the φ direction. Therefore, if the reference angle is 0 °, the arm 12 can rotate in the φ direction in the range of −30 ° to + 30 °. Of course, the rotatable range in the φ direction is not limited to ± 30 °.

  The arm 12 has a guide mechanism 15 which is a first guide mechanism. The guide mechanism 15 is disposed between the central portion of the arm 12 and one end. For example, the guide mechanism 15 is provided in the arm 12 by forming a guide groove in one surface of the arm 12. In the arm 12, a surface on which the guide mechanism 15 is formed is referred to as a guide forming surface 12a. For example, at the reference angle, the guide forming surface 12a is a plane parallel to the YZ plane. Of course, the guide forming surface 12a is not limited to a plane. In addition, the guide mechanism 15 may be formed by attaching a guide rail etc. to the guide formation surface 12a.

  The guide mechanism 15 guides the movement of the optical system unit 110. The optical system unit 110 is provided movably along the guide mechanism. For example, in the guide forming surface 12 a, the guide mechanism 15 is formed in an arc shape. The arc of the guide mechanism 15 is, for example, an arc centered on the vicinity of the observation center point O. Therefore, the optical system unit 110 moves along the arc on the guide forming surface 12a.

  As shown in FIG. 2, at the reference angle, the guide forming surface 12 a is offset from the Z axis in the −X direction. That is, at the reference angle, the plane including the guide forming surface 12a is arranged not to intersect the Z axis. Specifically, in accordance with the size of the optical system unit 110, the objective lens 120, etc., the guide forming surface 12a of the arm 12 is disposed offset from the Z axis.

  An objective lens 120 is attached to the sample side of the optical system unit 110. At the reference angle, the optical axis OX of the objective lens 120 is arranged to intersect the Z axis. The guide forming surface 12 a is disposed so as to be shifted in the −X direction from the observation center point O such that the optical axis OX of the objective lens 120 passes through the observation center point O. The objective lens 120 is disposed on the observation center point O side of the optical system unit 110. When the optical system unit 110 rotationally moves along the guide mechanism 15, the objective lens 120 rotationally moves in the θ direction together with the optical system unit 110. In addition, when the rotating shaft 11 rotates, the optical system unit 110 attached to the arm 12 and the objective lens 120 also rotate in the φ direction. Therefore, the optical system unit 110 and the objective lens 120 rotationally move in both directions θ and φ with the observation center point O as a rotation center point.

  The optical system unit 110 has, for example, a plurality of optical elements and a housing. The housing accommodates a plurality of optical elements. Therefore, along with the movement of the optical system unit 110, the plurality of optical elements disposed in the housing move together. The optical system unit 110 includes an optical system that propagates illumination light (illumination light) directed to the sample, and at least a portion of an optical system that propagates observation light from the sample to the light detector. Specifically, the optical system unit 110 includes a scanner or the like that scans the irradiation light irradiated to the sample. For example, the optical system unit 110 has a galvano mirror or the like as a scanner. Of course, the optical system unit 110 may have an optical element other than the scanner, for example, a mirror, a dichroic mirror, a light detector or the like. A specific example of the optical system provided in the optical system unit 110 will be described later.

  The illumination light scanned by the optical system unit 110 is incident on the objective lens 120. The objective lens 120 condenses the illumination light scanned by the optical system unit 110. The illumination light is condensed on the objective lens 120 toward the sample located at the observation center point O. The focal point of the illumination light by the objective lens 120 can be the observation center point O. If only the objective lens 120 is moved in the Y direction, it is also possible to focus on a spherical surface centered on the observation center point O. Also, when observation light from a sample is incident on the objective lens 120, the objective lens 120 refracts the observation light. Then, observation light from the sample is incident on the optical system unit 110 through the objective lens 120. The observation light is detected by a light detector provided in the optical system unit 110.

  When the optical system unit 110 is rotationally moved along the guide mechanism 15, the objective lens 120 is also rotationally moved (arrow θ in FIG. 1). That is, the objective lens 120 and the optical system unit 110 integrally rotate and move. The optical system unit 110 and the objective lens 120 are movably attached to the arm 12 by the guide mechanism 15. Here, as shown in FIG. 1, the moving direction of the optical system unit 110 along the guide mechanism 15 is the θ direction.

  At the reference angle in the φ direction, the θ direction is the rotation direction around the rotation axis parallel to the X axis. The θ direction changes according to the rotation angle of the rotation shaft 11 in the φ direction. The optical system unit 110 moves by ± 30 ° in the θ direction. That is, the guide mechanism 15 has a 60 ° arc. Of course, the movement range in the θ direction is not limited to ± 30 °. The rotation in the θ direction along the guide mechanism 15 is also referred to as θ axis rotation.

  Furthermore, a first weight 14 is attached to the arm 12. The first weight 14 is fixed to the guide forming surface 12 a of the arm 12. The first weight 14 is a counter weight provided to reduce fluctuation of φ rotation. The first weight 14 preferably has a weight equal to or greater than the total weight of the optical system unit 110 and the objective lens 120. The first weight 14 is, for example, disposed at a position symmetrical to the optical system unit 110 with the observation center point O interposed therebetween. Specifically, at the reference position, the optical system unit 110 and the first weight 14 are disposed at symmetrical positions with respect to the observation center point O.

  Furthermore, a second weight 13 is attached to the other end side of the rotating shaft 11. The rotating shaft 11 passes through the inside of the second weight 13. The second weight 13 is fixed to the rotary shaft 11 and is not fixed to the pedestal 17. The second weight 13 is provided so that the tip end side (arm 12 side) of the rotation shaft 11 does not incline downward. For example, the second weight 13 preferably has a weight equal to or greater than the total weight of the arm 12, the first weight 14, the optical system unit 110, and the objective lens 120.

  Here, when the φ axis is at the reference angle, a state in which the optical axis OX of the objective lens 120 is parallel to the Y axis is taken as a reference position in the θ direction. That is, when the rotation axis 11 is at the reference angle and the optical system unit 110 is at the reference position in the guide mechanism 15, the objective lens 120 is disposed directly above the observation center point O.

  Here, an operation in which the optical system unit 110 rotationally moves along the guide mechanism 15 will be described with reference to FIG. FIG. 3 is a YZ plan view schematically showing a configuration for explaining the operation along the guide mechanism 15. As shown in FIG. FIG. 3 shows the operation of the optical system unit 110 and the objective lens 120 at the reference angle of φ axis rotation.

  Furthermore, in FIG. 3, the optical system unit 110 at the reference position and the objective lens 120 are shown as an optical system unit 110 b and an objective lens 120 b. The optical system unit 110a and the objective lens 120a are rotationally moved in the + θ direction (clockwise in FIG. 3) from the reference position, and the optical system unit is rotationally moved in the −θ direction (counterclockwise in FIG. 3). The lens 110c is shown as an objective lens 120c.

  The guide mechanism 15 is provided in an arc shape centering on the observation center point O. Therefore, even when the optical system unit 110 and the objective lens 120 are rotationally moved in the θ direction, the optical axis OX of the objective lens 120 passes through the observation center point O. Not only the positions of the optical system unit 110 and the objective lens 120 but also the angles change according to the θ angle. For example, the optical axis OXb of the objective lens 120b is parallel to the Y axis, but the optical axis OXa of the objective lens 120a is inclined in the + θ direction from the Y axis. Further, the optical axis OXc of the objective lens 120c is inclined in the −θ direction from the Y axis.

  Therefore, the optical axis OX of the objective lens 120 passes through the observation center point O regardless of the θ angle of the optical system unit 110. The center of rotational movement of the optical system unit 110 in the θ direction is the observation center point O. Thus, the sample S at or near the observation center point O can be observed from various directions. In addition, if only the objective lens 120 is moved in the Y direction, it is also possible to focus on a spherical surface centered on the observation center point O.

  As described above, when the rotary shaft 11 rotates, the arm 12 rotates in the φ direction. Therefore, the optical system unit 110 and the objective lens 120 are also rotated in the φ direction together with the arm 12. Thereby, the objective lens 120 around the two axes and the optical system unit 110 can be rotated. Therefore, observation from various directions is possible in the two axial directions of the φ direction and the θ direction. The center of rotational movement of the optical system unit 110 in the φ direction is the observation center point O.

  Furthermore, the objective lens 120 is fixed to the optical system unit 110. Therefore, alignment of the optical system can be easily performed. That is, if incident light is introduced into the optical system unit 110 from an appropriate direction, the sample S can be illuminated. Thus, adjustment of the optical system can be facilitated.

  For example, in FIG. 3, illumination light is introduced to the optical system unit 110 by the optical fiber 26. That is, one end of the optical fiber 26 may be fixed to the entrance of the optical system unit 110. The optical fiber 26 may be bent so that tension is not applied even when the optical system unit 110 is rotationally moved in the φ direction and the θ direction. This enables observation from various directions with a simple configuration. Furthermore, observation from various directions is possible without moving the sample S.

Second Embodiment
The observation device 100 according to the second embodiment will be described with reference to FIG. FIG. 4 is a perspective view showing the configuration of the main part of the observation device 100. As shown in FIG. The present embodiment is different from the first embodiment in the configuration for introducing the irradiation light to the optical system unit 110. In the first embodiment, light is introduced to the optical system unit 110 by an optical fiber, but in the present embodiment, irradiation light is introduced to the optical system unit 110 using the mirror 103, the rotating mirror 104, and the movable mirror 105. doing. The basic configuration of the holding device 10 is the same as that of the first embodiment, and thus the description thereof is omitted. For example, the configuration and operation of the arm 12 and the rotary shaft 11 are the same as in the first embodiment.

  The mirror 103 and the rotating mirror 104 are disposed on the Z axis. The mirror 103 is fixed to, for example, a pedestal 17 (not shown in FIG. 4) shown in FIG. The rotating mirror 104 is fixed to the arm 12. The rotation shaft 11 is disposed between the mirror 103 and the rotation mirror 104. The rotating shaft 11 is hollow so that the irradiation light can pass through the inside of the rotating shaft 11.

  The irradiation light reflected by the mirror 103 passes through the inside of the rotating shaft 11 and enters the rotating mirror 104. The light reflected by the mirror 103 propagates along the Z axis. The rotating mirror 104 reflects the light emitted from the mirror 103 in the direction of the movable mirror 105. The irradiation light reflected by the rotating mirror 104 is reflected by the movable mirror 105. The irradiation light reflected by the movable mirror 105 enters the optical system unit 110. Then, as in the first embodiment, the irradiation light L1 illuminates the sample via the optical system unit 110 and the objective lens 120. An angle correction mirror 111 is provided inside the optical system unit 110.

  The rotating mirror 104 is fixed to the arm 12 or the rotating shaft 11. Here, the rotating mirror 104 is attached to the arm 12 on the Z axis. Therefore, when the rotating shaft 11 rotates, the angle of the rotating mirror 104 changes. That is, the rotating mirror 104 is rotatably provided so as to rotate with the rotating shaft 11. Since the rotating mirror 104 is disposed on the Z axis, the position of the rotating mirror 104 does not change but the direction changes due to the φ axis rotation. That is, the rotating mirror 104 rotates around the φ axis in place.

  The movable mirror 105 is movably attached to the arm 12. Specifically, the movable mirror 105 moves in accordance with the position of the optical system unit 110 in the θ direction. The irradiation light L1 reflected by the movable mirror 105 is incident on the optical system unit 110. The optical system unit 110 is provided with an angle correction mirror 111. The operation of the angle correction mirror 111 will be described later.

  The movable mirror 105 follows the movement so that the incident position of the irradiation light L1 to the angle correction mirror 111 becomes constant. Thereby, even when the position of the optical system unit 110 in the θ direction changes, the irradiation light L1 can be appropriately introduced to the optical system unit 110. Furthermore, the rotating mirror 104 and the movable mirror 105 rotate in the φ direction together with the arm 12. Therefore, the observation direction can be changed in the two axial directions of the φ direction and the θ direction.

  The operation of the movable mirror 105 interlocked with the rotational movement of the optical system unit 110 in the θ direction will be described with reference to FIG. FIG. 5 is a YZ plan view schematically showing the operation of the movable mirror 105 interlocked with the optical system unit 110. As shown in FIG.

  Similarly to FIG. 3, the optical system unit 110 at the reference position and the objective lens 120 are shown as an optical system unit 110 b and an objective lens 120 b. The movable mirror 105 corresponding to the optical system unit 110b is shown as the movable mirror 105b. The optical system unit 110a and the objective lens 120a are rotationally moved in the + θ direction from the reference position, and the optical system unit 110c and the objective lens 120c are rotationally moved in the −θ direction. The movable mirror 105 corresponding to the optical system unit 110a is shown as the movable mirror 105a, and the movable mirror 105 corresponding to the optical system unit 110c is shown as the movable mirror 105c.

  The arm 12 is provided with a linear guide mechanism 19 which is a second guide mechanism. Specifically, a guide plate 18 provided with a linear guide mechanism 19 is fixed to the arm 12. The movable mirror 105 is movably attached to the linear guide mechanism 19. The linear guide mechanism 19 guides the linear movement of the movable mirror 105. Specifically, the linear guide mechanism 19 is provided in parallel to the optical axis direction OX1 between the rotating mirror 104 and the movable mirror 105. Thus, the movable mirror 105 moves in a direction parallel to the optical axis OX1. Therefore, as the movable mirror 105 moves along the linear guide mechanism 19, the distance between the rotating mirror 104 and the movable mirror 105 changes.

  The position of the movable mirror 105 is changed according to the θ angle of the optical system unit 110. For example, in FIG. 5, the movable mirror 105b corresponding to the optical system unit 110b is at a higher position than the movable mirrors 105a and 105c corresponding to the optical system units 110a and 110c. That is, the operation of the movable mirror 105 is interlocked with the operation of the optical system unit 110 in the θ direction. Therefore, even when the optical system unit 110 rotationally moves in the θ direction, the irradiation light L 1 reflected by the movable mirror 105 is incident on the optical system unit 110.

  Note that the incident angle of the irradiation light L1 to the optical system unit 110 changes in accordance with the θ angle. That is, although the irradiation light L1 reflected by the movable mirror 105 propagates in parallel with the Z axis, the inclination angle of the optical system unit 110 changes according to the position of the optical system unit 110 in the θ direction. For example, although the objective lens 120 b is parallel to the Y axis, the objective lens 120 a and the objective lens 120 c are inclined from the Y axis. Therefore, the optical system unit 110 has the angle correction mirror 111. The inclination of the angle correction mirror 111 changes in accordance with the θ angle. Thereby, the irradiation light L1 can propagate the inside of the optical system unit 110 appropriately. The operation of the angle correction mirror 111 will be described later.

  In the present embodiment, light is incident on the optical system unit 110 using the mirror 103, the rotating mirror 104, and the movable mirror 105. In the present embodiment, light propagates in space using the mirror 103, the rotating mirror 104, and the movable mirror 105. That is, light propagates in the air and is incident on the optical system unit 110.

  For example, when the pulse laser light propagates in the optical fiber 26 as in the first embodiment, the pulse length becomes longer according to the propagation optical path length in the optical fiber 31. On the other hand, in the present embodiment, since the mirror 103, the rotating mirror 104, and the movable mirror 105 are used, the irradiation light can propagate in the air. Thereby, it can be prevented that the pulse length of the irradiation light becomes long. Therefore, in the second embodiment, it is preferable to use pulsed laser light as irradiation light. In particular, the observation apparatus 100 according to the present embodiment is suitable for a two-photon excitation microscope or a multiphoton excitation microscope using short pulse light such as femtosecond laser light as excitation light.

(Configuration of optical system)
Next, the configuration of the optical system of the observation apparatus 100 will be described with reference to FIG. FIG. 6 is a view showing the configuration of the optical system of the observation apparatus 100. As shown in FIG. In the present embodiment, the observation device 100 is described as a two-photon excitation microscope, but the observation device 100 is not limited to a two-photon excitation microscope. For example, the observation apparatus 100 may be a scanning microscope or a confocal microscope.

  The observation apparatus 100 includes a light source 101, a beam expander 102, a mirror 103, a rotating mirror 104, a movable mirror 105, an optical system unit 110, and an objective lens 120. The optical system unit 110 includes an angle correction mirror 111, a mirror 112, a mirror 113, a first scanner 114, a second scanner 115, a scan lens 116, a tube lens 117, a dichroic mirror 118, and a relay. A lens 131, an infrared cut filter 132, a dichroic mirror 133, a light detector 134, and a light detector 135 are provided. Angle correction mirror 111, mirror 112, mirror 113, first scanner 114, second scanner 115, scan lens 116, tube lens 117, dichroic mirror 118, relay lens 131, infrared The cut filter 132, the dichroic mirror 133, the light detector 134, and the light detector 135 are accommodated in the housing of the optical system unit 110.

  First, the configuration of the illumination optical system for irradiating the sample S with the irradiation light L1 from the light source 101 will be described. The light source 101 generates illumination light. The light source 101 is, for example, a pulse laser light source. Specifically, the light source 101 is a femtosecond mode-locked titanium-sapphire laser and generates short-pulse near-infrared light. Here, the pulse width of the irradiation light is 100 fsec, the repetition frequency is 80 MHz, and the average power is 3 W. By using the short pulse laser light, it is possible to increase the peak power and to reduce the average power. Therefore, damage to the sample S can be reduced, which is suitable for observation of a biological sample. The light source 101 is variable in wavelength, and the variable wavelength range is 690 to 1040 nm. The irradiation light from the light source 101 is a parallel luminous flux. Of course, the light source 101 is not limited to a titanium sapphire laser.

  The irradiation light from the light source 101 enters the beam expander 102. The beam expander 102 has a pair of lenses and expands the spot diameter of the irradiation light. Specifically, the beam expander 102 expands the beam diameter of the irradiation light so that the beam diameter corresponds to the area of the first scanner 114 and the second scanner 115. The irradiation light from the beam expander 102 is reflected by the mirror 103, the rotating mirror 104, and the movable mirror 105. As described above, the rotation mirror 104 rotates about the φ axis by the rotation of the rotation shaft 11. The movable mirror 105 moves in accordance with the rotation angle around the θ axis.

The irradiation light reflected by the movable mirror 105 is introduced into the optical system unit 110. The irradiation light L1 incident on the optical system unit 110 is incident on the angle correction mirror 111. The irradiation light L1 reflected by the angle correction mirror 111 is incident on the mirror 112. The angle correction mirror 111 is
The first angle variable mirror provided in the optical system unit 110 is provided. The angle correction mirror 111 is a rotating mirror provided rotatably so as to change the reflection angle of light. The inclination of the angle correction mirror 111 changes in accordance with the θ angle. The rotation angle of the angle correction mirror 111 changes in accordance with the position of the optical system unit 110 in the guide mechanism 15. That is, the angle correction mirror 111 operates to follow so that the incident position of the irradiation light l1 to the mirror 112 becomes constant. Thus, even when the optical system unit 110 rotationally moves in the θ direction, the inclination and the incident position of the irradiation light L1 with respect to the mirror 112 can be made constant.

  The incident light L 1 reflected by the angle correction mirror 111 is reflected by the mirrors 112 and 113 and enters the first scanner 114. The irradiation light L 1 scanned by the first scanner 114 is incident on the second scanner 115. The first scanner 114 and the second scanner 115 are optical scanners using MEMS (Micro Electro Mechanical systems), galvano mirrors, and the like. As the first scanner 114, a resonant scanning mirror can be used. As the second scanner 115, a servo type scanning mirror such as a MEMS mirror operated by servo control can be used.

  On the sample S, the first scanner 114 deflects the irradiation light L1 in the Xs direction (not shown), and the second scanner 115 deflects the irradiation light L1 in the Ys direction (not shown). Here, the Xs direction and the Ys direction are orthogonal to each other in the sample S. That is, in the plane orthogonal to the optical axis OX of the objective lens 120, the Xs direction and the Ys direction are orthogonal to each other. Therefore, on the sample S, two-dimensional scanning in the Xs direction and the Ys direction is performed. Thereby, in the sample S, a predetermined area can be observed. In addition, it does not specifically limit about the structure of the optical scanner which scans irradiation light, A well-known optical scanner can be used. For example, a biaxial scanning mirror may be used.

  The irradiation light scanned by the first scanner 114 and the second scanner 115 enters the scan lens 116. The scan lens 116 condenses the irradiation light on the primary image plane of the objective lens 120. At the primary image plane, the deflection angles of the first scanner 114 and the second scanner 115 scan the position of the irradiation light.

  The irradiation light that has passed through the scan lens 116 enters the tube lens 117 after passing through the primary image plane. The tube lens 117 is disposed between the scan lens 116 and the objective lens 120. The tube lens 117 refracts the irradiation light into a parallel light flux. The illumination light from the tube lens 117 enters the dichroic mirror 118. The dichroic mirror 118 transmits infrared light and reflects visible light. Therefore, the illumination light passes through the dichroic mirror 118 and enters the objective lens 120. Thus, the irradiation light L1 from the tube lens 117 is incident on the objective lens 120 via the dichroic mirror 118.

  The objective lens 120 condenses the irradiation light to illuminate the sample S. That is, the irradiation light that has passed through the objective lens 120 is irradiated onto the sample S as a minute spot. When the irradiation light is incident on the minute spot of the sample S, the fluorescent substance in the sample S is excited. Since only the fluorescence by two-photon excitation is observed with the irradiation light as infrared light, fluorescence is generated only from the fluorescent substance of the focused spot of the irradiation light. In other words, almost no fluorescence is generated from other than the focal plane of the irradiation light. The focal point of the objective lens 120 is an observation point. This enables observation at high resolution (resolution).

  Further, since the irradiation position of the irradiation light L1 is two-dimensionally scanned by the first scanner 114 and the second scanner 115, a predetermined area of the sample can be illuminated. A two-dimensional image can be constructed by detecting the fluorescence when scanning the irradiation light L1.

  The sample S is placed on a sample stage (not shown). The sample stage is disposed so as not to interfere with the arm 12 and the like. Further, by changing the distance between the sample S on the sample stage and the objective lens 120, the focal position of the irradiation light with respect to the sample S can be moved in the optical axis direction. By moving the objective lens 120 relative to the sample S along the optical axis direction, observation at different depths becomes possible. By focusing on the deep part of the sample S, it is possible to observe the deep part of the sample S. For example, the objective lens 120 may be provided movably with respect to the optical system unit 110. Alternatively, the sample stage may be a drive stage. Further, by setting the sample stage as a three-dimensional drive stage, it is possible to move the observation position on the sample S to an arbitrary position. further,

  The illumination light collected by the objective lens 120 is incident on the sample S. Then, at the focal point of the objective lens 120, fluorescence is generated by two-photon excitation. That is, when one fluorescent molecule of the sample S absorbs two photons almost at the same time and becomes an excited state, fluorescence is emitted when returning to a stable state. The fluorescence generated by such two-photon excitation is used as observation light. The wavelength of fluorescence is different depending on the fluorescent substance.

  Next, a detection optical system through which observation light propagates will be described. The observation light is emitted from the illuminated minute spot in all directions. Therefore, a part of the observation light generated by the sample S becomes return light returning to the objective lens 120. The observation light which has been returned light enters the objective lens 120. The objective lens 120 refracts the incident observation light. Here, the objective lens 120 refracts the return light (observation light) from the sample S so as to be a parallel light flux. The observation light having passed through the objective lens 120 is incident on the dichroic mirror 118. As described above, the dichroic mirror 118 transmits infrared light and reflects visible light. Therefore, the fluorescence (observation light) generated by two-photon excitation is visible light. Therefore, the observation light is reflected in the direction of the relay lens 131 by the dichroic mirror 118. The irradiation light which is incident on the sample S and reflected as it is is also incident on the dichroic mirror 118 through the objective lens 120. However, since the irradiation light is infrared light, it passes through the dichroic mirror 118. Thereby, it is possible to prevent the detection of the irradiation light that becomes noise.

  As described above, the dichroic mirror 118 serves as a light separating unit that separates the irradiation light from the objective lens 120 and the observation light according to the wavelength. Also, the dichroic mirror 118 is disposed between the objective lens 120 and the tube lens 117. Therefore, the dichroic mirror 118 separates the observation light from the objective lens 120 from the illumination light before the observation light is descanned by the first scanner 114 and the second scanner 115. Thus, the observation apparatus 100 which becomes a two-photon microscope constitutes a non-descanned detector.

  The observation light reflected by the dichroic mirror 118 is incident on the relay lens 131. The relay lens 131 condenses the observation light. The infrared cut filter 132 is a filter that cuts infrared light and transmits visible light. By disposing the infrared cut filter 132 in the optical path, it is possible to prevent infrared light (irradiation light) which is noise from being detected by the light detectors 134 and 135.

  The observation light having passed through the infrared cut filter 132 is incident on the dichroic mirror 133. The dichroic mirror 133 branches the observation light according to the wavelength. The dichroic mirror 133 reflects green light and transmits red light. The green observation light reflected by the dichroic mirror 133 is detected by the light detector 134. The red observation light transmitted through the dichroic mirror 133 is detected by the light detector 135.

  The photodetectors 134 and 135 are, for example, photomultipliers (photomultipliers), and output detection signals according to the detected light amounts. For example, the photodetectors 134 and 135 are composed of one large light receiving pixel. Specifically, the detection signal is input to a control device described later. The control device stores the scanning position of the irradiation light and the detection signal in association with each other. Furthermore, the control device stores the detected light amount in association with the θ angle and the φ angle. That is, the position in the sample S, the observation direction, and the detected light amount are associated with each other.

  In the above description, two systems of photodetectors 134 and 135 are used because multiple simultaneous staining is performed in which fluorescent substances (fluorescent dyes) having different fluorescence wavelengths are simultaneously excited at one wavelength. That is, two systems of detection optical systems are provided to simultaneously observe different fluorescent substances. However, the detection optical system may be only one system. That is, a system of detection optical systems corresponding to the fluorescence wavelength of the fluorescent substance to be observed may be provided. In this case, the dichroic mirror 133 light detector 134 and the like become unnecessary. Furthermore, the number of detection optical systems may be three or more.

  In the observation device 100, infrared light is used as the irradiation light. Therefore, the irradiation light L1 reaches the deep portion of the sample S. Therefore, it is suitable for three-dimensional observation which observes the deep part of sample S. In particular, when the sample S is a living body, since absorption of infrared light is small in living tissue, it is suitable for observing a deep portion of the sample S.

  Here, the operation of the movable mirror 105 and the angle correction mirror 111 will be described with reference to FIG. FIG. 7 is a diagram showing the configuration of the optical system of the observation apparatus 100. As shown in FIG. FIG. 7 differs from FIG. 6 in the θ angle. The basic configuration of the optical system is the same as that shown in FIG.

  When the θ angle changes, the position of the movable mirror 105 changes. That is, the distance between the rotating mirror 104 and the movable mirror 105 changes in accordance with the θ angle. Thus, even when the optical system unit 110 rotationally moves in the θ direction, the irradiation light L1 enters the optical system unit 110.

  Furthermore, when the θ angle changes, the angle of the angle correction mirror 111 changes. Thus, even when the tilt of the optical system unit 110 changes due to the rotational movement in the θ direction, the irradiation light L1 appropriately propagates along the optical axis of the optical system. Therefore, even when the θ angle changes, illumination can be performed with the irradiation light L1 of which the alignment is guaranteed.

  FIG. 8 is a diagram for explaining the relationship between the θ angle of the optical system unit 110, the position of the movable mirror 105, and the tilt angle of the angle correction mirror 111. As shown in FIG. Note that FIG. 8 shows the internal configuration of the optical system unit 110 in a simplified manner. For example, some optical elements such as the dichroic mirror 118 are omitted.

  FIG. 8 shows a configuration in which the rotation axis 11 is at a reference angle. Further, θ angle = 0 ° when the optical axis OX of the objective lens 120 is parallel to the Y axis. In the case of θ angle = 0 °, the optical system unit 110 is at the reference position. In the case of θ angle = 0 °, the angle of the reflection surface of the angle correction mirror 111 is inclined 45 ° with respect to the Y axis. When the θ angle = 0 °, the distance between the Z axis (φ axis) and the movable mirror 105 is L.

As shown in FIG. 8, the θ angle when the optical system unit 110 is rotationally moved around the θ axis is θ _a . The optical element in the case of θa is illustrated with the symbol a. For example, in FIG. 8, the objective lens in the case where the θ angle is the reference position (0 °) is the objective lens 120, and the objective lens in the case of θ angle = θa is the objective lens 120a. If theta angle of theta _a, the angle of the reflecting surface of the angle correction mirror 111a is 45 ° + θ _a / 2. Assuming that the movement amount of the movable mirror 105 when the θ angle is θ _a is δ, then δ = L (1−cos θ _a ). Thus, the sample S can be appropriately illuminated by driving the angle correction mirror 111 in accordance with the θ angle.

  The observation apparatus 100 according to the first embodiment has a configuration in which the beam expander 102, the mirror 103, the rotating mirror 104, and the movable mirror 105 are replaced with an optical fiber 26. If the incident angle of the irradiation light L1 entering the angle correction mirror 111 from the optical fiber 26 is constant, the rotation operation of the angle correction mirror 111 is unnecessary.

(Control system of observation apparatus 100)
Next, the configuration of the control system of the observation apparatus 100 will be described using FIG. The observation apparatus 100 includes a mirror drive motor 141, a θ axis motor 145, a θ axis sensor 146, a φ axis motor 147, a φ axis sensor 148, a chiller 151, a laser power source 152, a current / voltage converter 161, a protection circuit 162, and a high voltage power source 163. , An A / D converter 164, a current-voltage converter 166, a protection circuit 167, a high voltage power supply 168, an A / D converter 169, an interface (I / F) 171, and a control device 172. In FIG. 9, a part of optical elements of the observation device 100 is omitted.

  The I / F 171 is responsible for input and output between each device and the control device 172. The control device 172 controls each device such as a motor and a power supply. Each motor is, for example, a driving mechanism such as a stepping motor or a servomotor. The controller 172 outputs control signals for controlling the respective motors. Therefore, the controller 172 can appropriately control the operation of each motor. Of course, the drive mechanism may be other than a motor. The control device 172 is, for example, a processing device such as a workstation or a personal computer. For example, the control device 172 includes a processor, a memory, and the like. Each device can be controlled by the processor executing a computer program stored in the memory.

  The chiller 151 cools the light source 101. The laser power supply 152 supplies power to the light source 101. The power supplied from the laser power supply 152 is controlled by the controller 172. The control device 172 also controls the first scanner 114 and the second scanner 115.

  The φ-axis motor 147 is a rotary shaft drive mechanism for rotationally driving the rotary shaft 11 around the φ-axis. The φ-axis motor 147 is controlled by the controller 172. For example, when the user inputs a direction to be observed, the φ-axis sensor 148 is an angle sensor that detects the rotation angle (φ angle) of the rotating shaft 11 by the φ-axis motor 147. The φ-axis sensor 148 is, for example, a motor encoder. The φ-axis sensor 148 outputs a sensor signal corresponding to the detected φ angle to the controller 172.

  The θ-axis motor 145 is a unit drive mechanism for rotationally moving the optical system unit 110 around the observation center point O. The θ-axis motor 145 rotationally moves the optical system unit 110 along the guide mechanism 15. The θ-axis motor 145 is controlled by the controller 172. For example, when the user inputs a direction to be observed, the control device 172 generates a drive signal for driving the θ-axis motor 145 and the φ-axis motor 147.

  The θ axis sensor 146 is a sensor that detects the position or the θ angle of the optical system unit 110 by the θ axis motor 145. The θ-axis sensor 146 is, for example, a motor encoder. The θ-axis sensor 146 outputs a sensor signal corresponding to the detected position or angle to the controller 172. The control device 172 controls the θ-axis motor 145 and the φ-axis motor 147 so that the θ-angle and the φ-angle become desired angles according to a command from the user.

  The mirror drive motor 141 is a mirror drive mechanism that moves the movable mirror 105. The mirror drive motor 141 changes the distance between the rotating mirror 104 and the movable mirror 105 in accordance with the position of the optical system unit 110 in the guide mechanism 15. A mirror drive motor 141 moves the movable mirror 105 along the linear guide mechanism 19. The controller 172 controls the mirror drive motor 141 based on the detection signal from the θ-axis sensor 146. Thereby, the movable mirror 105 operates in conjunction with the optical system unit 110. The irradiation light L1 can be introduced into the optical system unit 110.

  Further, the control device 172 controls the angle correction mirror 111 based on the detection signal from the θ-axis sensor 146. The angle correction mirror 111 operates in conjunction with the optical system unit 110. The irradiation light L1 can be propagated in an appropriate direction. Thus, the control device 172 controls the mirror drive motor 141 and the angle correction mirror 111 according to the θ angle.

  The detection signal output from the light detector 134 is current-voltage converted by the current-voltage converter 161. The current-voltage converter 161 converts the current into a voltage by the load resistance. As a result, the voltage of the detection signal indicates the amount of detected light. The current-voltage converter 161 outputs the detection signal subjected to current-voltage conversion to the A / D converter 164. Then, an A / D (Analog to Digital) converter 164 A / D converts the detection signal. Then, a detection signal that has become a digital signal is input to the control device 172. The controller 172 stores the detection signal, that is, the detection signal. The light detector 134 is supplied with power generated by the high voltage power supply 163. In addition, a protection circuit 162 is provided between the high voltage power supply 163 and the current voltage converter 161.

  The detection signal output from the photodetector 135 is current-voltage converted by the current-voltage converter 166. As a result, the voltage of the detection signal indicates the amount of detected light. The current-voltage converter 166 outputs the current-voltage converted detection signal to the A / D converter 169. Then, an A / D (Analog to Digital) converter 169 A / D converts the detection signal. Then, a detection signal that has become a digital signal is input to the control device 172. The controller 172 stores the detection signal, that is, the detected light amount. The power generated by the high voltage power supply 168 is supplied to the light detector 135. Further, a protection circuit 167 is provided between the high voltage power supply 168 and the current voltage converter 166.

  Then, the control device 172 stores the scanning position of the irradiation light L1 by the first scanner 114 and the second scanner 115 in association with the detected light amount. Further, the control device 172 stores the detected light amount in association with the θ angle and the φ angle. By doing this, the sample S can be observed from various directions.

Third Embodiment
The operation of the optical system unit 110 in the observation apparatus according to the third embodiment will be described with reference to FIG. In the present embodiment, the observation device has a configuration in which the optical system unit operates without the need to measure the θ angle by the θ-axis sensor 146. Specifically, the observation device does not have the θ-axis sensor 146, and the movable mirror 105 is replaced with the servo mirror 205. Further, the angle correction mirror 111 and the mirror 112 provided inside the optical system unit 110 are replaced with the first position sensor mirror 211 and the second position sensor mirror 212. The other configuration is the same as in the first and second embodiments, and thus the description is omitted.

  The servo galvano mirror 205 changes the reflection angle of the irradiation light L1 according to the θ angle. That is, the servo galvano mirror 205 is a second variable angle mirror provided on the arm 12. The servo galvano mirror 205 is, for example, a rotating mirror provided rotatably so as to change the reflection angle of light. The servo galvano mirror 205 reflects the irradiation light L1 from the rotating mirror 104 toward the optical system unit 110.

  The first position sensor mirror 211 changes the reflection angle of the irradiation light L1 according to the θ angle. The first position sensor mirror 211 is a third variable-angle mirror provided in the optical system unit 110. The first position sensor mirror 211 is a rotating mirror provided rotatably so as to change the reflection angle of light. The first position sensor mirror 211 reflects the irradiation light L1 reflected by the servo galvano mirror 205. The second position sensor mirror 212 is a fixed mirror fixed in the optical system unit 110. The second position sensor mirror 212 reflects the irradiation light L1 reflected by the first position sensor mirror 211.

  The first position sensor mirror 211 and the second position sensor mirror 212 have a sensor that detects the incident position of the irradiation light. Specifically, the second position sensor mirror 212 has a position sensor that detects the incident position of the irradiation light on the second position sensor mirror 212. Then, the second position sensor mirror 212 outputs a position detection signal according to the detected position to the first position sensor mirror 211. The servo galvano mirror 205 is rotated in accordance with the incident position of the irradiation light L 1 to the first position sensor mirror 211. The first position sensor mirror 211 is rotated in accordance with the incident position of the irradiation light L1 to the second position sensor mirror 212.

  The first position sensor mirror 211 changes the reflection angle based on the position detection signal from the second position sensor mirror 212. The second position sensor mirror 212 has a position sensor that detects the incident position of the irradiation light to the second position sensor mirror 212. The first position sensor mirror 211 is rotated following the incident position of the irradiation light L1 to the second position sensor mirror 212. For example, the first position sensor mirror 211 changes the reflection angle at which the irradiation light is reflected so that the irradiation light is incident on the predetermined position of the second position sensor mirror 212.

  Similarly, the first position sensor mirror 211 has a position sensor that detects the incident position of the irradiation light on the first position sensor mirror 211. Then, the first position sensor mirror 211 outputs a position detection signal according to the detected position to the servo galvano mirror 205.

  The servo galvano mirror 205 changes the reflection angle based on the position detection signal from the first position sensor mirror 211. The servo galvano mirror 205 is rotated following the incident position of the irradiation light L1 to the first position sensor mirror 211. For example, the servo galvano mirror 205 changes the reflection angle at which the irradiation light is reflected so that the irradiation light is incident on the predetermined position of the first position sensor mirror 211. Thus, regardless of the θ angle, the irradiation light L1 is incident on the centers of the first position sensor mirror 211 and the second position sensor mirror 212.

  The configuration of the first position sensor mirror 211 will be described with reference to FIG. FIG. 11 is a diagram for explaining the configuration of the first position sensor mirror 211. As shown in FIG. Since the configuration of the second position sensor mirror 212 is the same as the configuration of the first position sensor mirror 211, the description will be omitted. That is, the configurations of the first position sensor mirror 211 and the second position sensor mirror 212 are the same.

  The first position sensor mirror 211 includes a substrate 221, photodetectors 222 and 223, and a reflective film 224. The substrate 221 is a transparent glass substrate or the like. A reflective film 224 is formed on the front side of the substrate 221 (the incident side of the irradiation light L1). On the back side of the substrate 221, photodetectors 222 and 223 are formed.

  The reflective film 224 is a film that transmits a part of the irradiation light L1. Therefore, a part of the irradiation light L1 passes through the reflective film 224 and the substrate 221, and enters the light detectors 222 and 223. In addition, the reflection film 224 reflects most of the irradiation light L1 toward the second position detection mirror 212.

  The light detector 222 and the light detector 223 are provided on the back side of the substrate 221. The light detector 222 and the light detector 223 take the irradiation light L1 transmitted through the substrate 221 and the reflection film 224. The light detector 222 and the light detector 223 serve as position sensors for detecting the incident position of the irradiation light L1. Specifically, the light detector 222 and the light detector 223 constitute a two-part photodiode. Each of the light detectors 222 and 223 is a photodiode, and outputs a current corresponding to the amount of detected light as a detection signal. For example, the boundary between the light detector 222 and the light detector 223 passes through the center of the first position detection mirror 211. The servo galvano mirror 205 is subjected to tracking control so that the center of the incident position of the irradiation light L1 is on the boundary between the light detector 222 and the light detector 223. A servo loop is formed so that the detected light amounts of the light detector 222 and the light detector 223 are equal.

  Specifically, when the optical system unit 110 rotates by θ, the first position sensor mirror 211 moves in the direction perpendicular to the boundary between the light detector 222 and the light detector 223. The rotation of the servo galvano mirror 205 is controlled such that the incident position of the irradiation light L1 follows the boundary between the light detector 222 and the light detector 223. By using such a servo loop, the θ-axis sensor 146 of the optical system unit 110 becomes unnecessary.

  An example of the configuration for realizing the above-described servo loop will be described with reference to FIG. As shown in FIG. 12, the light detector 222 of the first position sensor mirror 211 outputs a detection signal to the current-voltage converter 261. The photodetector 223 of the first position sensor mirror 211 outputs a detection signal to the current-voltage converter 262. The current-voltage converter 261 performs current-voltage conversion on the detection signal from the light detector 222. The current-voltage converter 262 current-voltage converts the detection signal from the light detector 223. Thus, the voltage of the detection signal indicates the amount of detected light.

  The current voltage converter 261 and the current voltage converter 262 output the detection signal to the differential amplifier 263. Therefore, when the detection signals of the current-voltage converter 261 and the current-voltage converter 262 match, the output of the differential amplifier 263 becomes 0V. When the detection signal of the current-voltage converter 261 is larger than the detection light amount of the current-voltage converter 262, the output of the differential amplifier 263 becomes positive (plus). When the detection signal of the current-voltage converter 261 is smaller than the detection light amount of the current-voltage converter 262, the output of the differential amplifier 263 becomes negative (minus).

  The servo galvano mirror 205 is controlled to follow so that the output of the differential amplifier 263 becomes 0V. Thus, even when the optical system unit 110 rotates by θ, the irradiation light L1 is controlled to be incident on the center of the first position sensor mirror 211. The servo galvano mirror 205 is rotated in accordance with the detection position of the irradiation light L 1 by the first position sensor mirror 211.

  Similar servo loops are formed for the first position sensor mirror 211 and the second position sensor mirror 212. The light detector 222 of the second position sensor mirror 212 outputs a detection signal to the current-voltage converter 271. The photodetector 223 of the second position sensor mirror 212 outputs a detection signal to the current-voltage converter 272. The current-voltage converter 271 performs current-voltage conversion on the detection signal from the photodetector 223. The current-voltage converter 272 performs current-voltage conversion on the detection signal from the light detector 223. Thus, the voltage of the detection signal indicates the amount of detected light.

  The current-voltage converter 271 and the current-voltage converter 272 output the detection signal to the differential amplifier 273. Therefore, when the detection signals of the current-voltage converter 271 and the current-voltage converter 272 match, the output of the differential amplifier 273 becomes 0V. When the detection signal of the current-voltage converter 271 is larger than the detection light amount of the current-voltage converter 272, the output of the differential amplifier 273 becomes positive (plus). When the detection signal of the current-voltage converter 271 is smaller than the detection light amount of the current-voltage converter 272, the output of the differential amplifier 273 becomes negative (minus).

  The first position sensor mirror 211 is controlled to follow so that the output of the differential amplifier 273 becomes 0V. Thus, even when the optical system unit 110 rotates by θ, the propagation direction of the irradiation light L1 reflected by the second position sensor mirror 212 becomes constant. The first position sensor mirror 211 is rotated in accordance with the detection position of the irradiation light L 1 by the second position sensor mirror 212. That is, regardless of the θ angle, the irradiation light L1 enters the first scanner 114 at a constant angle. As a result, the first scanner 114 can scan the irradiation light L1 for which the alignment is guaranteed.

Modification 1
The configuration of a modification of the third embodiment will be described using FIG. FIG. 13 is a diagram for illustrating follow-up control with respect to the θ angle. In the first modification, follow-up control for the θ angle is performed without using special mirrors such as the first position sensor mirror 211 and the second position sensor mirror 212. In addition, about the content which overlaps with Embodiment 1-3, description is abbreviate | omitted suitably.

  In the first modification, the optical system unit 110 includes a tracking mirror 260, a current-voltage converter 261, a current-voltage converter 262, a differential amplifier 263, a first position sensor 265, a first beam sampler 266, and a mirror 270. A current-voltage converter 271, a current-voltage converter 272, a differential amplifier 273, a second position sensor 275, and a second beam sampler 276 are provided.

  That is, the first position sensor mirror 211 is replaced by the follow-up mirror 260, and the second position sensor mirror 212 is replaced by the mirror 270. Furthermore, a first beam sampler 266 is disposed between the tracking mirror 260 and the mirror 270. A second beam sampler 276 is disposed downstream of the mirror 270.

  The current-voltage converter 261, the current-voltage converter 262, the differential amplifier 263, the current-voltage converter 271, the current-voltage converter 272, and the differential amplifier 273 are the same as those in FIG. Further, each of the first position sensor 265 and the second position sensor 275 is a two-split photodiode. Therefore, like the light detectors 222 and 223, the first position sensor 265 and the second position sensor 275 can respectively detect the incident position of the irradiation light L1.

  The following mirror 260 is disposed between the servo galvano mirror 205 and the mirror 270. The follow-up mirror 260 is a third variable-angle mirror provided in the optical system unit 110. The following mirror 260 is a rotating mirror provided rotatably so as to change the reflection angle of light. The mirror 270 is a fixed mirror fixed in the optical system unit 110.

  The first beam sampler 266 is disposed in the optical path between the tracking mirror 260 and the mirror 270 and at a position close to the tracking mirror 260. The first beam sampler 266 extracts a part of the irradiation light L1 reflected by the tracking mirror 260. The irradiation light L 1 extracted by the first beam sampler 266 is incident on the first position sensor 265. The servo galvano mirror 205 is rotated in accordance with the incident position detected by the first position sensor 265. Therefore, it is possible to form a servo loop that performs tracking control so that the irradiation light L1 is incident on the center of the first position sensor 265.

  The second beam sampler 276 extracts a part of the irradiation light L1 reflected by the mirror 270. The optical path between the mirror 270 and the first scanner 114 (not shown in FIG. 13) is located close to the mirror 270. The irradiation light L 1 extracted by the second beam sampler 276 is incident on the second position sensor 275. The tracking mirror 260 is rotated in accordance with the incident position detected by the second position sensor 275. Therefore, it is possible to form a servo loop that performs tracking control so that the irradiation light L1 is incident on the center of the second position sensor 275.

  With such a configuration, the same effect as the configuration of FIG. 11 can be obtained. In addition, since the loss of light by the beam sampler is about several percent, there is no particular problem if the light amount of the irradiation light L1 is sufficient.

  Thus, according to the observation apparatus 100 concerning this Embodiment 1-3, a sample can be observed from various directions. Also, it is possible to observe from various directions without moving the sample. For this reason, the observation apparatus 100 is suitable for brain researches such as monkeys and mice.

  In the above description, the first to third angle variable mirrors are described as rotating mirrors that rotate around an axis parallel to the reflecting surface, but one or more of the first to third angle variable mirrors are It may be a mirror whose angle changes without rotation. For example, as the angle variable mirror, a steering mirror, a MEMS mirror, or the like can be used instead of the rotating mirror. That is, the angle variable mirror may be any mirror that can change the direction of reflected light. For example, in the variable-angle mirror, the angle of the reflective surface changes in accordance with the control signal from the controller 172. Thereby, the reflection angle of light changes.

  In addition, by using the variable angle mirror and the four-divided sensor (S-shaped sensor) in combination, it is possible to correct the declination angle of the other element (other direction) not associated with the change of the θ angle. As the other elements, for example, it is possible to correct an angle of deflection due to deflection when the arm 12 is inclined in the φ direction, an optical axis deviation generated when tuning the laser light source, and the like. In this case, as the angle variable mirror, it is preferable to use an angle variable mirror capable of two-axis operation such as a steering mirror. Then, the position of the irradiation light is detected using a 4-division photodiode which is a 4-division sensor. Then, feedback control may be performed so that the center of the irradiation light coincides with the center of the four-divided photodiode. The four-divided sensor can be configured by a light detector disposed on the back side of the substrate as shown in FIGS. In this case, the irradiation light transmitted through the reflective film and the substrate is incident on the four-divided sensor. Alternatively, as shown in FIG. 13, the beam sampler may be disposed in the optical path so that the four-divided sensor detects the irradiation light L1 extracted by the beam sampler.

  As mentioned above, although the invention made by the present inventor was concretely explained based on an embodiment, the present invention is not limited to the embodiment mentioned already, A various change in the range which does not deviate from the gist It goes without saying that it is possible.

DESCRIPTION OF SYMBOLS 10 holding apparatus 11 rotating shaft 12 arm 12a guide formation surface 13 1st weight 14 2nd weight 15 guide mechanism 16 bearing 17 base 18 guide plate 19 linear guide mechanism 100 observation apparatus 101 light source 102 beam expander 103 mirror 104 rotating mirror 105 movable mirror 110 optical system unit 111 angle correction mirror 112 mirror 113 mirror 114 first scanner 116 second scanner 116 scan lens 117 tube lens 118 dichroic mirror 120 objective lens 131 relay lens 132 infrared cut filter 133 dichroic mirror 134 light Detector 135 Photodetector 141 Mirror drive motor 145 θ-axis motor 146 θ-axis sensor 147 φ-axis motor 148 φ-axis sensor 15 Reference Signs List 1 chiller 152 laser power supply 161 current voltage converter 162 protection circuit 163 high voltage power supply 164 A / D converter 166 current voltage converter 167 protection circuit 168 high voltage power supply 169 A / D converter 171 interface 172 control device O observation center point OX light axis S sample

Claims (8)

A rotation axis rotatably provided about the first axis;
A rotary shaft drive mechanism for rotating the rotary shaft around the first shaft;
An arm attached to the rotation shaft, the arm provided with a first guide mechanism;
An optical system unit movably provided along the first guide mechanism, the optical system unit having a scanner for scanning irradiation light irradiated to the sample.
An objective lens attached to the sample side of the optical system unit and focusing the irradiation light scanned by the scanner onto the sample;
A unit drive mechanism for moving the optical system unit along the first guide mechanism such that the objective lens is rotationally moved about a rotation center point on an extension of the first axis A device ,
The rotating shaft is hollow so that light can pass through the inside of the rotating shaft,
The observation device
A rotating mirror attached to the arm on the first axis and reflecting illumination light that has passed through the inside of the rotation axis;
A movable mirror disposed movably with respect to the arm and reflecting light emitted from the rotating mirror toward the optical system unit;
And a mirror driving mechanism for moving the movable mirror so as to change a distance between the rotating mirror and the movable mirror according to a position of the optical system unit in the first guide mechanism. The observation apparatus according to claim 1 , wherein the arm is provided with a second guide mechanism that guides the rectilinear movement of the movable mirror by the mirror drive mechanism. The optical system unit comprises: a first variable-angle mirror on which the irradiation light reflected by the movable mirror is incident;
The illumination light reflected by the first variable angle mirror is scanned by the scanner,
Wherein the angle of the first variable angle mirror, the observation apparatus according to claim 1 or 2 changes according to the position of the optical system unit in the first guide mechanism. A rotation axis rotatably provided about the first axis;
A rotary shaft drive mechanism for rotating the rotary shaft around the first shaft;
An arm attached to the rotation shaft, the arm provided with a first guide mechanism;
An optical system unit movably provided along the first guide mechanism, the optical system unit having a scanner for scanning irradiation light irradiated to the sample.
An objective lens attached to the sample side of the optical system unit and focusing the irradiation light scanned by the scanner onto the sample;
A unit drive mechanism for moving the optical system unit along the first guide mechanism such that the objective lens is rotationally moved about a rotation center point on an extension of the first axis A device,
The rotating shaft is hollow so that light can pass through the inside of the rotating shaft,
The observation device
A rotating mirror attached to the arm on the first axis and reflecting illumination light that has passed through the inside of the rotation axis;
And a second variable-angle mirror provided on the arm and reflecting light emitted from the rotating mirror toward the optical system unit,
The optical system unit is
A third angle variable mirror attached to the optical system unit and reflecting illumination light reflected by the second angle variable mirror;
A mirror that reflects the illumination light reflected by the third variable angle mirror;
The angle of the third variable-angle mirror changes in accordance with the incident position of the irradiation light to the mirror,
The third according to the incident position of the irradiation light with respect to the angle variable mirror, observation device angle of the second variable angle mirror you change. The third variable angle mirror and the mirror each have a substrate,
A reflective film provided on the front side of the substrate and formed to transmit a part of the irradiation light;
A position sensor provided on the back side of the substrate to detect an incident position of the irradiation light;
The angle of the third variable-angle mirror changes in accordance with the incident position detected by the position sensor of the mirror,
The observation apparatus according to claim 4 , wherein an angle of the second variable-angle mirror changes in accordance with an incident position detected by the position sensor of the third variable-angle mirror. A first beam sampler disposed in an optical path between the third variable angle mirror and the mirror and extracting a part of the irradiation light reflected by the third variable angle mirror;
A first position sensor for detecting an incident position of the irradiation light extracted by the first beam sampler;
A second beam sampler disposed in a light path between the mirror and the scanner for extracting a portion of the irradiation light reflected by the mirror;
And a second position sensor for detecting an incident position of the irradiation light extracted by the first beam sampler,
The angle of the second variable-angle mirror changes in accordance with the incident position detected by the first position sensor,
The observation apparatus according to claim 4 , wherein an angle of the third variable angle mirror changes in accordance with an incident position detected by the second position sensor. The observation apparatus according to any one of claims 1 to 6 , further comprising a pulse laser light source that generates the irradiation light. The observation apparatus according to any one of claims 1 to 7 , wherein a counter weight is attached to the arm at a position symmetrical to the optical system unit with the rotation center point interposed therebetween.

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